Vibrio cholerae, a major human pathogen responsible for both endemic and epidemic cholera, has an absolute requirement for iron. Because V. cholerae must be able to obtain iron in a variety of different environments in and outside of host organisms, it has evolved multiple iron acquisition systems and can use iron from a variety of sources. These iron transport systems include the endogenous siderophore vibriobactin, receptors for exogenous siderophores and for heme, and ferric and ferrous iron uptake systems. Iron transport systems are tightly regulated to avoid either iron starvation or iron toxicity, and there is both transcriptional and post-transcriptional regulation of iron transport and iron metabolism. Regulation is mediated by iron and the global regulator Fur and by the small RNA RyhB. There is evidence for additional levels of regulation. We will characterize the V. cholerae iron transport systems, the regulation of their expression, and their role in survival of V. cholerae in the environment and in the host. These systems will be characterized using molecular biology, genetics, and biochemical techniques. As we complete the characterization of all the iron transport systems that this pathogen uses, we can determine their roles in pathogenesis and further dissect the complex regulatory network by which environmental signals regulate the expression V. cholerae iron transport genes. The specific aims of this grant are: 1) Identify the complete complement of V. cholerae iron transport systems. 2) Characterize the ferrous iron transporter Feo and the novel VciB iron acquisition system. These systems are of particular interest since they are both expressed by bacteria during infection of the host and are likely to be major iron uptake systems in the microaerobic environment of the intestine. 3) Determine the mechanism of regulation of V. cholerae iron transport genes in response to environmental signals. The systems respond to multiple, different signals to optimize iron acquisition and growth of V. cholerae in the variety of environments in which it is found, and we propose to define these regulatory networks. It is important to understand bacterial iron transport systems and their regulation, because the ability of pathogens to compete with their host for this essential element is a critical component of the host-pathogen interaction. Defining and characterizing the V. cholerae iron transport systems at the molecular level and understanding their expression in response to different environmental conditions will provide the basis for developing strategies to control this major human pathogen. The results of these studies are likely to be broadly applicable to human pathogenic bacteria, since the majority of them must acquire iron during infection.